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[Cancer Research 66, 527-534, January 1, 2006]
© 2006 American Association for Cancer Research

Immunology

Subversion of Protein Kinase C{alpha} Signaling in Hematopoietic Progenitor Cells Results in the Generation of a B-Cell Chronic Lymphocytic Leukemia–Like Population In vivo

Rinako Nakagawa1, Jae Won Soh3 and Alison M. Michie1,2

1 Division of Immunology, Infection and Inflammation and 2 Division of Cancer Science and Molecular Pathology, Section of Experimental Haematology, University of Glasgow, Scotland, United Kingdom and 3 Laboratory of Signal Transduction, Department of Chemistry, Inha University, Incheon, Korea

Requests for reprints: Alison M. Michie, Division of Immunology, Infection and Inflammation, Western Infirmary, University of Glasgow, Scotland G11 6NT, United Kingdom. Phone: 44-141-211-2161; Fax: 44-141-337-3217; E-mail: a.michie{at}udcf.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived mature B cells with the distinctive phenotype CD19hi CD5+ CD23+ IgMlo, which are refractory to apoptosis. An increased level of apoptosis has been observed on treatment of human B-CLL cells with protein kinase C (PKC) inhibitors, suggesting that this family of protein kinases mediate survival signals within B-CLL cells. Therefore, to investigate the ability of individual PKC isoforms to transform developing B cells, we stably expressed plasmids encoding PKC mutants in fetal liver–derived hematopoietic progenitor cells (HPC) from wild-type mice and then cultured them in B-cell generation systems in vitro and in vivo. Surprisingly, we noted that expression of a plasmid-encoding dominant-negative PKC{alpha} (PKC{alpha}-KR) in HPCs and subsequent culture both in vitro and in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity over untransfected cells and phenotypically resemble human B-CLL cells. In the absence of growth factors and stroma, these B-CLL-like cells undergo cell cycle arrest and, consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 expression. These studies therefore identify a unique oncogenic trigger for the development of a B-CLL-like disease resulting from the subversion of PKC{alpha} signaling. Our findings uncover novel avenues not only for the study of the induction of leukemic B cells but also for the development of therapeutic drugs to combat PKC{alpha}-regulated transformation events. (Cancer Res 2006; 66(1): 527-34)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in the Western world and can be characterized by an accumulation of long-lived mature B cells with the distinctive phenotype CD19hi CD5+ CD23+ IgMlo, which are refractory to apoptosis and have undergone cell cycle arrest in the G0-G1 phase (1). A variety of mechanisms have been shown to account for the clonal expansion of this leukemic population of B cells, including chromosomal abnormalities and intrinsic defects in their apoptosis machinery due to both higher levels of the antiapoptotic protein Bcl-2 family member proteins and aberrations of the p53 tumor suppressor gene (1, 2). Notably, treatment of human B-CLL cells with protein kinase C (PKC) and phosphatidylinositol 3-kinase inhibitors leads to increased levels of apoptosis (3–6), suggesting that these protein kinases may mediate survival signals within B-CLL cells.

PKC represents a family of closely related serine/threonine protein kinases that share structural features and requirements for Ca2+, phospholipid, and diacylglycerol (7). PKCs are divided into three subfamilies, classic Ca2+-dependent isoforms ({alpha}, ßI/II, {gamma}), novel Ca2+-independent isoforms ({delta}, {varepsilon}, {eta}, {theta}), and atypical isoforms ({zeta},{lambda}/{iota}; ref. 7). PKC regulates key biological events, including proliferation, differentiation, cell survival, and gene transcription in response to diverse stimuli (8). To execute these cellular processes, PKC is regulated by, and functionally interacts with, several proto-oncogenes, such as phosphoinositide-dependent kinase 1, Ras, Raf-1, and Bcl-10, which are key regulators of Akt/protein kinase B, mitogen-activated protein kinase, and nuclear factor-{kappa}B signaling pathways involved in the survival and proliferation of B cells (9–13). Therefore, it is perhaps unsurprising that dysregulation of specific PKC isoforms, particularly PKC{alpha}, have been implicated in a diverse range of cancers (14). Indeed, PKC{alpha} has become a focus for potential therapies with the use of antisense oligonucleotides, small interfering RNA, and PKC inhibitors in a range of cancer types (6, 15–17).

In an effort to address the potential role of specific PKC isoforms during B-cell transformation, we stably expressed PKC mutants in fetal liver–derived hematopoietic progenitor cells (HPC) from wild-type mice using retrovirally mediated techniques and then tested the ability of these constructs to transform developing B lymphocytes (18, 19). Our studies reveal that disruption of PKC{alpha} signaling by the expression a dominant-negative PKC{alpha} construct in HPCs results in the spontaneous development of a B-CLL-like population both in vitro and in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, plasmids, and cell lines. ICR mice were purchased from Harlan-Olac (Oxon, United Kingdom) and maintained at the University of Glasgow Central Research Facilities (Glasgow, United Kingdom). Recombinase activating gene-1–deficient (RAG-1–/–) mice were bred and maintained in-house (20). Timed-pregnant mice were generated, and liver was extracted from fetuses at day 14 of gestation. All animals were maintained in accordance with local and home office regulations. Retroviral constructs were generated by subcloning the gene of interest (dominant-negative PKC isoforms) into the retroviral backbone (MIEV), 5' of the internal ribosomal entry site, allowing the bicistronic expression of the gene of interest and green fluorescent protein (GFP; ref. 21). The dominant-negative PKC isoforms were generated by introducing a point mutation at the lysine in the ATP-binding domain of each PKC isoform as follows: PKC{alpha}, PKC{alpha}-KR(K368R) and PKC{delta}, PKC{delta}-KR(K376R) (18, 22). A constitutively active form of PKC{alpha} (PKC{alpha}-CAT) was generated by deleting the regulatory NH2-terminal domain of PKC{alpha} (18). A hemagglutinin (HA) tag was engineered onto the COOH-terminal of each PKC construct to allow confirmation of expression at the protein level using an anti-HA antibody (18). The retroviral vectors were stably expressed in the retroviral packaging line, GP+E.86 (21, 23). OP9 is a bone marrow stromal cell line derived from op/op mice, which are deficient in macrophage colony-stimulating factor (M-CSF; ref. 19).

Retroviral infection of HPC-enriched cell populations. HPC-enriched fetal liver cells derived from wild-type ICR mice were prepared as described previously (24) and cocultured with the mitomycin C–treated retroviral packaging line in complete medium [high-glucose DMEM containing 13% fetal bovine serum (BioWhittaker UK Ltd., Wokingham, United Kingdom), 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 10 mmol/L HEPES, 50 µmol/L 2-mercaptoethanol (2-ME), and 10 µg/mL gentamicin (Invitrogen, Paisley, United Kingdom)] supplemented with 500 µg/mL polybrene (Sigma-Aldrich Co., Poole, United Kingdom) and 10 ng/mL cytokines [interleukin (IL)-6, IL-7, and stem cell factor (SCF); PeproTech EC Ltd., London, United Kingdom] for 24 to 48 before being placed in either an in vitro B-cell generation system or adoptively transferred into RAG-1–/– mice.

B-cell generation in vitro. To analyze the ability of retrovirally infected HPC populations to develop into mature B lymphocytes in vitro, the cells were removed from the retroviral packaging lines and cocultured with OP9 cells (19) in the presence of IL-6, IL-7, and SCF (10 ng/mL of each; 20,000 cells per well of a six-well plate). The developing cells were removed from the OP9 layer by gentle pipetting and placed onto a fresh layer of OP9 cells in the presence of 10 ng/mL IL-7 only once every 5 to 7 days. Cells were removed for analysis as indicated in the figure legends.

Flow cytometric analysis. R-phycoerythrin (PE)-conjugated, allophycocyanin (APC)-conjugated, and biotin-conjugated anti-mouse antibodies were used for flow cytometric analysis (BD Biosciences, Oxford, United Kingdom). Cell samples were prepared for fluorescence-activated cell sorting (FACS) analysis as described previously (24). The cells were acquired on a FACSCalibur flow cytometer using the CellQuest software package (BD Biosciences) to acquire and the FlowJo software package (Tree Star, Inc., Stanford, CA) to analyze the data. All data shown are live gated by size and lack of propidium iodide (PI; Sigma-Aldrich) uptake.

PKC activity assay. Cell samples were washed in ice-cold PBS twice and resuspended in ice-cold sample preparation buffer [30 mmol/L Tris-HCl (pH 7.0), 15 mmol/L NaCl, 5 mmol/L EGTA, 2.6 mmol/L EDTA, 25 mmol/L 2-ME, and 0.05% (v/v) Triton X-100 supplemented with Complete Protease Inhibitor Cocktail (Roche Diagnostics Ltd., Lewes, United Kingdom)]. Lysates were sonicated for 5 seconds five times and then centrifuged at 15,000 x g for 60 minutes at 4°C. The supernatant was then collected and the protein concentration was determined using Bradford method (25). Protein (10 µg) was assayed per sample in triplicate. PKC activity was measured based on the phosphorylation of a PKC substrate peptide using a nonradioactive protein kinase assay according to the manufacturer's protocol (Calbiochem-Novabiochem Co., Darmstadt, Germany).

SDS-PAGE and Western blotting. Cell samples were removed from the OP9 cell line and either treated with phorbol ester [20 or 200 nmol/L 12-O-tetradecanoylphorbol-13-acetate (TPA); Cell Signaling Technology, Hitchin, United Kingdom] for 30 minutes or left unstimulated as indicated in the figure legends. Thereafter, cell lysates (4 µg/sample) were prepared, resolved, and transferred onto polyvinylidene difluoride membranes as described previously (24). The level of phosphorylation of PKC substrates was detected using an anti-phosphoserine PKC substrate antibody (Cell Signaling Technology) followed by horseradish peroxidase–conjugated goat anti-rabbit IgG (Cell Signaling Technology). Blots were revealed using SuperSignal West Pico Chemiluminescent Substrate (Perbio, Tattenhall, United Kingdom; ref. 24).

Cell cycle analysis. Cell samples were removed from the OP9 cell line 2 days before PI analysis and the dead cells were removed by density centrifugation over Lymphocyte-Mammal (VH Bio Ltd., Newcastle, United Kingdom) as described previously (24). The cells were then resuspended in complete medium in the conditions described in the figure legends at a concentration of 2 x 105 per well. Cellular DNA content was detected by PI staining. Cells were lysed in 0.2% Triton X-100-PBS solution and incubated for 10 minutes on ice. The lysed cells were treated with 0.5% RNase (Sigma-Aldrich) to remove RNA followed by the addition of PI to a final concentration of 50 µg/mL (26). PI incorporation was analyzed using a FACSCalibur flow cytometer using the CellQuest software package, with an activated doublet discriminator module, to acquire and the FlowJo software package to analyze the data.

Bromodeoxyuridine incorporation. Cell samples were removed from the OP9 cell line 2 days before analysis and the dead cells were removed by density centrifugation. The resultant cells were resuspended in complete medium in the conditions described in the figure legends at a concentration of 2 x 105 per well. Bromodeoxyuridine (BrdUrd; 50 µmol/L) was added 23 hours before cell harvesting, at which time the cells were washed once in PBS and then fixed using the Cytofix/Cytoperm kit as described by the manufacturer (BD Biosciences). To expose incorporated BrdUrd, cells were washed in BD Perm/Wash solution and treated with DNase at the final concentration of 300 µg/mL for 1 hour at 37°C and then stained with anti-BrdUrd-APC antibody (1:300 dilution; BD Biosciences) for 20 minutes at 4°C. Cells were then resuspended in 7-actinomycin D to stain DNA and washed in the BD Perm/Wash solution, and the samples were acquired on a FACSCalibur flow cytometer using the CellQuest software package. All data shown were size gated by discrimination based on forward scatter and side scatter. FlowJo software package was used to analyze the data.

RNA purification and reverse transcription-PCR. RNA was isolated from HPC-OP9 cocultures throughout the culture period as indicated in the figure legends using the RNA-Easy RNA purification procedure (Qiagen, Crawley, United Kingdom). Semiquantitative PCR reactions were done using the same serially diluted cDNA batches shown for ß2-microglobulin (ß2m; ref. 24). All PCR products correspond to the expected molecular sizes. Gene-specific primers used for PCR are as follows: ;ß2m_U: 5'-GGCGTCAACAATGCTGCTTCT-3' and ß2m_L: 5'-CTTTCTGTGTTTCCCGCTCCC-3' (277 bp), Bcl-2_U: 5'-GGGAGAACAGGGTATGA-3' and Bcl-2_L: 5'-ATCTCTGCGAAGTCACG-3' (323 bp), and Bcl-XL_U: 5'-GAAGCAGAGAGGGAGACC-3' and Bcl-XL_L: 5'-CTCCTTGTCTACGCTTTC-3' (351 bp).

Adoptive transfer of retrovirally infected HPCs into mice. To analyze the ability of retrovirally infected HPC populations to develop into mature lymphocytes in vivo, the cells were removed from the retroviral packaging lines, and after removal of the dead and nonlymphoid cells by density centrifugation over Lymphocyte-Mammal at 600 x g for 15 minutes at room temperature, the cells were resuspended in PBS at a concentration of 1 x 107/mL and injected i.p. into neonatal RAG-1–/– mice at a concentration of 1 x 106. The mice were sacrificed 2 to 5 weeks postinjection and the bone marrow, spleen, lymph node, and tumors were removed for analysis as indicated in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HPCs exhibit a proliferative advantage on subversion of PKC{alpha} signaling. To determine the potential role of individual PKC isoforms to transform developing B cells, fetal liver–derived HPCs were prepared from wild-type mice and retrovirally infected with a plasmid encoding either vector control (MIEV) or dominant-negative PKC{alpha} (PKC{alpha}-KR) or PKC{delta} (PKC{delta}-KR) constructs (18) and then cocultured the cells with the bone marrow stromal cell line OP9 in the presence of growth factors, thus promoting the generation of B lymphocytes in vitro (19). The retroviral vector (MIEV) enables the generation of a bicistronic message encoding the gene of interest (dominant-negative PKC isoform) coupled to GFP. Therefore, on stable integration into mouse fetal liver–derived HPCs, the retrovirally infected cells can be monitored using flow cytometry (21).

Flow cytometric analysis of fetal liver HPC cultures immediately after retroviral infection revealed a GFP+ population within each retroviral condition (Fig. 1A; day 2: MIEV 25%, PKC{alpha}-KR 4.2%, and PKC{delta}-KR 11%). Of note, a greater proportion of cells were retrovirally infected in the MIEV control coculture (Fig. 1A; day 2). This is likely due to the absence of a gene of interest in the control vector 5' of GFP. As the OP9-HPC coculture progressed, it became clear that PKC{alpha}-KR-expressing (GFP+) cells displayed a growth advantage over the untransduced (GFP-) cells as shown by a 20-fold increase in the percentage of GFP+ cells from days 2 to 14 (4.2% versus 98%; Fig. 1A). This was in contrast to cocultures containing HPCs retrovirally infected with either MIEV or PKC{delta}-KR, which maintained a relatively constant percentage of GFP expression (Fig. 1A). The observed growth advantage of PKC{alpha}-KR-expressing GFP+ cells was accompanied by substantial elevation in cell numbers compared with MIEV- and PKC{delta}-KR-expressing cocultures. Indeed, PKC{alpha}-KR-expressing cells proliferate 1.5-fold more in a 24-hour period than MIEV-expressing cells. PI analysis confirmed that the relative elevation in cell number in PKC{alpha}-KR cocultures was not due to an increase in apoptosis in MIEV cocultures (data not shown).



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  		Figure 1. Subversion of PKC{alpha} signals in HPCs results in an enhanced proliferative capacity of developing cells. HPC-enriched fetal liver cells were prepared from wild-type mice and retrovirally infected with either vector alone (MIEV) or dominant-negative PKC{alpha} (PKC{alpha}-KR) or PKC{delta} (PKC{delta}-KR). A, retrovirally infected cells were seeded onto OP9 cells in the presence of growth factors (SCF, IL-6, and IL-7) and cultured for up to 3 weeks. During this period, the cultures were sampled and analyzed by flow cytometry for relative amounts of GFP expression (%). Histogram plots were live and size gated. RCN, relative cell number. Representative of at least five experiments. B, MIEV and PKC{alpha}-KR retrovirally infected cells were seeded on OP9 cells in the presence of growth factors and cultured for 12 days. Then, the cells were harvested by Ficoll density gradient centrifugation and cultured on OP9 cells in the presence of growth factors for 2 days. BrdUrd (BrdU) solution was added before flow cytometric analysis. Cells were size gated before analysis of % BrdUrd incorporation. Columns, average % BrdUrd incorporated cells of three separate experiments; bars, SE. *, P < 0.05, MIEV over PKC{alpha}-KR.

 
To address whether the elevation in cell number in PKC{alpha}-KR (GFP+)–expressing cells was due to an enhanced proliferation, the cells were analyzed by BrdUrd incorporation to measure the level of DNA synthesis in the cell cultures (Fig. 1B). This set of analysis was carried out on MIEV and PKC{alpha}-KR retrovirally infected HPCs after 12 days of culture as >90% of PKC{alpha}-KR-expressing coculture were GFP+ at this stage (Fig. 1A; data not shown). It was noted that PKC{alpha}-KR-expressing cells possessed an increased proliferative capacity as shown by their ability to incorporate significantly more BrdUrd compared with MIEV-expressing cells (Fig. 1B). Taken together, these results show that in the presence of stroma and growth factors the subversion of PKC{alpha} signals leads to an increase in the proliferative capacity of developing HPCs.

PKC{alpha}-KR-expressing cells display a decrease in PKC activity. To confirm that PKC{alpha}-KR and PKC{delta}-KR expression could be correlated with GFP expression, we made use of the fact that a HA tag was fused to the COOH terminus of the PKC-KR constructs during the construction of the vector, thus allowing confirmation of expression of the exogenously added constructs at the protein level (18). Flow cytometric analysis revealed that HA expression within each PKC-KR-expressing GP+E.86 packaging cell line correlated with the level of GFP expression, whereas the control MIEV-expressing cell line expressed high levels of GFP and background levels of HA (Fig. 2A). As expected, intracellular staining revealed that HPCs retrovirally infected with PKC{alpha}-KR exhibited a high expression of HA compared with MIEV control cells (Fig. 2B).



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  		Figure 2. PKC{alpha}-KR-expressing cells display a decrease in PKC activity. A, cell suspensions of MIEV, PKC{alpha}-KR, and PKC{delta}-KR-GP+E.86 retroviral packaging lines were analyzed by flow cytometry for the expression of GFP or fixed, permeabilized, and stained with an anti-HA antibody, which reveals the retrovirally introduced PKC{alpha}-KR or PKC{delta}-KR construct by virtue of the HA tag engineered to the 5' end of the construct. B, HPC-enriched fetal liver cells were prepared from wild-type mice and retrovirally infected with MIEV or PKC{alpha}-KR and then seeded on OP9 cells in the presence of growth factors and cultured for 16 days. PKC{alpha}-KR expression was determined by fixing and permeabilizing the cells, before staining with an anti-HA antibody, to reveal the retrovirally introduced PKC{alpha}-KR construct. C, lysates were prepared from MIEV, PKC{alpha}-KR, and PKC{alpha}-CAT-GP+E.86 retroviral packaging lines and PKC activity was assessed. Columns, % kinase activity relative to MIEV-GP+E.86 control cells (100%); bars, SE. D, lysates were prepared from MIEV and PKC{alpha}-KR retrovirally infected cells that were cultured as described above, before the determination of PKC activity. Columns, % kinase activity relative to MIEV fetal liver control cells (100%); bars, SE. E, MIEV and PKC{alpha}-KR retrovirally infected cells cultured as described above were incubated in the presence or absence of phorbol ester (0, 20, or 200 nmol/L TPA as indicated). Lysates were prepared and resolved by SDS-PAGE under reducing conditions and subsequently immunoblotted with a specific antibody against phosphoserine PKC substrates.

 
An attenuation of PKC activity was observed in PKC{alpha}-KR-expressing cells compared with MIEV cells in both GP+E.86 packaging cell lines (Fig. 2C) and HPC-OP9 cocultured cells (Fig. 2D). Indeed, a 50% reduction in the kinase activity was observed in HPCs retrovirally infected with PKC{alpha}-KR compared with MIEV-expressing cells, showing a significant decrease in PKC activity in these cells (Fig. 2D). Importantly, analysis of the kinase activity of a GP+E.86 packaging line stably expressing an established constitutively active form of PKC{alpha} mutant (PKC{alpha}-CAT; refs. 18, 21, 24) revealed an 34% elevation in kinase activity relative to MIEV-GP+E.86 cells (Fig. 2C). In support of these findings, immunoblotting analysis revealed a decrease in the level of serine phosphorylation of downstream substrates of PKC in lysates derived from PKC{alpha}-KR-expressing HPCs compared with MIEV-expressing HPCs (Fig. 2E). In addition, a reduction in the level of phosphorylation of PKC substrates was noted on TPA stimulation in PKC{alpha}-KR-expressing HPCs compared with MIEV control cells (Fig. 2E). Taken together, these results show that an attenuation of PKC{alpha} signals leads to an increase in the proliferative capacity of developing HPCs.

Disruption of PKC{alpha} signaling results in the outgrowth of a B-CLL-like population in vitro. To define the phenotype of the retrovirally infected populations, flow cytometric analysis of the GFP+ cells was carried out using cell surface markers that can define the developmental stages of B-cell maturation. As expected, >95% of the GFP+ cocultured cells expressed the B-cell markers CD45R (B220) and CD19, establishing that the retrovirally infected cells had committed to the B-cell lineage (Fig. 3; data not shown). However, closer phenotypic analysis of the cocultures revealed that although MIEV- and PKC{delta}-KR-expressing cells contained similar populations of normal developing B cells (CD19+ CD23- IgM+ CD5- CD45R+; Fig. 3), PKC{alpha}-KR-expressing cells had up-regulated CD19, CD23, and CD5 while expressing low levels of IgM. Indeed, analysis of the mean fluorescence intensity (MFI) of key cell surface markers on B lineage cells in MIEV compared with PKC{alpha}-KR-GFP+ populations revealed an up-regulation of CD19 (271 versus 476), CD23 (1.1 versus 14), and CD5 (5.1 versus 50) expression, whereas IgM expression was down-regulated on PKC{alpha}-KR (GFP+)–expressing B lineage cells (19 versus 2.8). These findings indicate that stable expression of PKC{alpha}-KR leads to an outgrowth of cells that phenotypically resemble human B-CLL (CD19hi CD5+ CD23+ IgMlo; Fig. 3; ref. 1). In addition to the established phenotype of human B-CLL cells observed in PKC{alpha}-KR-expressing cultures, we also noted an elevation in CD2 levels compared with MIEV- or PKC{delta}-KR-expressing cultures, thus broadening the B-CLL-like phenotype of the proliferating population in our in vitro mouse model system (Fig. 3). Molecular analysis of the immunoglobulin heavy chain locus in both MIEV and PKC{alpha}-KR cellular populations revealed that a similar pattern of multiple, random rearrangements occurred in both populations (data not shown; ref. 27). These results served to confirm that the proliferating PKC{alpha}-KR population arose from several distinct transformed progenitor cells. Therefore, these findings indicate that subversion of PKC{alpha} signaling in HPCs stimulates aberrant expansion of an oligoclonal population of developing B lymphocytes that resemble B-CLL.



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  		Figure 3. Subversion of PKC{alpha} signals results in the generation of a B-CLL-like population in vitro. HPC-enriched fetal liver cells were prepared from wild-type mice and retrovirally infected with MIEV, PKC{alpha}-KR, or PKC{delta}-KR. The retrovirally infected cells were then seeded on OP9 cells in the presence of growth factors and cultured for 10 days. The cultures were harvested and phenotypically characterized by flow cytometry: CD23 versus CD19, IgM versus {kappa} light chain, and CD5 versus CD2. Contour plots show profiles that are live and size and GFP+ gated; percentages are indicated. Representative of at least five experiments.

 
Subversion of PKC{alpha} signaling enables cells to evade apoptosis. One of the hallmarks of B-CLL is the accumulation of B lymphocytes that are arrested in the G0-G1 phase of the cell cycle and are refractory to apoptosis (1, 28). Therefore, to test whether the B-CLL-like cells were able to survive in the absence of both stroma and growth factors, we cultured MIEV or PKC{alpha}-KR retrovirally infected cells in the absence of both IL-7 and OP9 after 12 days in culture. Our results show that B lineage cells expressing PKC{alpha}-KR were able to survive under these conditions, whereas the majority of control (MIEV) cells were dead after 3 days in such cultures (Fig. 4A). These results established that the B-CLL-like cells generated on expression of PKC{alpha}-KR displayed a survival advantage over control cells. This finding was confirmed by carrying out PI analysis, which revealed that >3-fold more cells died in the control cell culture on withdrawal of stroma and growth factors compared with PKC{alpha}-KR-expressing cells, indicating that the cells were able to evade apoptosis (Fig. 4B). Moreover, the majority of PKC{alpha}-KR-expressing cells were halted in the G0-G1 phase of the cell cycle, a hallmark of B-CLL in vivo (Fig. 4B).



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  		Figure 4. Subversion of PKC{alpha} signals enable cells to survive in the absence of stromal cells and growth factors. HPC-enriched fetal liver cells were prepared from wild-type mice and retrovirally infected with either MIEV or PKC{alpha}-KR. A, MIEV and PKC{alpha}-KR retrovirally infected cells were seeded on OP9 cells in the presence of growth factors and cultured for 12 days. Then, the cells were harvested by Ficoll density gradient centrifugation and cultured in the absence of both OP9 cells and growth factors (day 0; triplicate wells of a 24-well plate for each day of counting). Cells were then counted on the following 3 consecutive days by flow cytometer. Columns, mean number of GFP+ cells of three separate experiments; bars, SE. Cells were live and size gated. ***, P < 0.0001, MIEV versus PKC{alpha}-KR. B, MIEV and PKC{alpha}-KR retrovirally infected cells were seeded on OP9 cells in the presence of growth factors and cultured for 12 days. Then, the cells were harvested by Ficoll density gradient centrifugation and cultured in the absence of both OP9 cells and growth factors for 2 days before PI analysis. Top, average ± SE percentage of cells at the indicated cell cycle stage taken from four separate experiments. ***, P < 0.0005, MIEV versus PKC{alpha}-KR; ** P < 0.005, MIEV versus PKC{alpha}-KR. Bottom, histogram of DNA content versus RCN is a representative of these experiments. White histogram, MIEV; gray histogram, PKC{alpha}-KR. The cellular data were size gated. C, MIEV and PKC{alpha}-KR retrovirally infected cells were seeded on OP9 cells in the presence of growth factors. Cells were removed at days 3, 8, and 14 of coculture and RNA/cDNA was prepared from the samples. Reverse transcription-PCR was done with ß2m, Bcl-2, and Bcl-xL primers. When normalized to ß2m expression, the expression levels of Bcl-2 and Bcl-xL in PKC{alpha}-KR-derived samples are in the range of 3.2- to 4.0-fold and 0.9- to 1.2-fold that of MIEV-derived samples, respectively, at day 14.

 
Bcl-2 has long been reported to be a causative factor in the increased survival rate of B-CLL cells (1, 29). Therefore, to gain an understanding of the mechanism by which the B-CLL-like population modulates apoptotic/survival rates, we analyzed the expression levels of the antiapoptotic factors Bcl-2 and Bcl-XL in control and PKC{alpha}-KR-expressing cells during in vitro cultures (1, 29). This analysis revealed that although Bcl-2 transcript levels were down-regulated at day 14 in control cultures they remained elevated in PKC{alpha}-KR-expressing cells, a characteristic of B-CLL (Fig. 4C). Indeed, when normalized to ß2m expression, the expression levels of Bcl-2 were ~4-fold higher than that of MIEV-derived samples at day 14 (Fig. 4C). In contrast, the level of Bcl-XL transcripts remained unchanged in both control and PKC{alpha}-KR-expressing cultures (Fig. 4C). Collectively, these studies provide a causal link between the subversion of PKC{alpha} signaling in developing B lymphocytes and the generation a transformed population of cells resembling human B-CLL that can escape apoptosis via a process that likely involves the up-regulation Bcl-2.

Inhibition of PKC{alpha} signaling in HPCs results in the generation of a B-CLL-like population in vivo. To test the impact of the stable expression of PKC{alpha}-KR in vivo, retrovirally infected fetal liver HPCs were adoptively transferred into RAG-1–/– mice, which lack mature B and T lymphocytes (20). The ability of wild-type donor cells to reconstitute lymphoid organs of host RAG-1–/– mice was assessed by analyzing the spleen and lymph nodes 2 to 4 weeks postinjection. Notably, flow cytometric analysis of spleen and lymph node samples revealed an increase in the percentage of GFP+ cells in PKC{alpha}-KR-HPC-injected mice compared with MIEV-HPC-injected mice, although the percentage of GFP+ cells in the PKC{alpha}-KR sample before injection was significantly less than that in the MIEV sample (Table 1; data not shown). This suggests that PKC{alpha}-KR-expressing cells display an elevated proliferative potential in vivo and supports our in vitro data (Fig. 1). This notion was supported by the finding that a reduction in the percentage of GFP+ HPCs in the MIEV sample before injection resulted in a reduction in the percentage of GFP+ cells recovered from the reconstituted spleen (Table 1).


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  		Table 1. HPCs display a proliferative advantage on subversion of PKC{alpha} signaling in vivo

 
Phenotypic analysis revealed that, as expected, a population of mature B lymphocytes was present in the GFP– populations of both MIEV- and PKC{alpha}-KR-injected mice (Fig. 5; CD19+ IgM+ IgD+ CD23+), indicating that the fetal liver cells derived from wild-type mice reconstituted RAG–/– mice successfully. Flow cytometric analysis of a spleen from an unreconstituted, age-matched RAG–/– mouse (no donor) highlights the presence of host-derived CD45R+ CD19+ IgM– IgD– CD23– cells, which are also present in the GFP– population of MIEV- and PKC{alpha}-KR-HPC donor reconstituted mice (Fig. 5). Mice injected with MIEV-HPCs possess a significant elevation in the percentage of IgM+ and IgD+ B lymphocytes in the GFP+ population, establishing that this is a successful method of generating mature B lymphocytes in vivo (Fig. 5). Interestingly, phenotypic analysis of GFP+ cells present in PKC{alpha}-KR-HPC-injected mouse spleen and lymph node revealed a population of cells that resembled the B-CLL-like cells that were generated in the in vitro cultures (IgMlo CD23+ CD5+ CD19hi IgDlo; Fig. 5; data not shown). Similar to that observed in the in vitro cocultures, analysis of the MFI of B-CLL cell surface markers on B lineage cells in MIEV compared with PKC{alpha}-KR-GFP+ populations revealed an up-regulation of CD19 (105 versus 144) and CD23 (47.5 versus 215) expression, whereas IgM expression was down-regulated on PKC{alpha}-KR (GFP+)–expressing B lineage cells (372 versus 33.5).



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  		Figure 5. A B-CLL-like population develops in vivo on injection of RAG-1–/– mice with PKC{alpha}-KR retrovirally infected HPCs. HPC-enriched fetal liver cells were prepared from wild-type mice and retrovirally infected with either MIEV or PKC{alpha}-KR. The retrovirally infected wild-type mouse-derived cells were injected i.p. into neonatal RAG-1–/– mice (1 x 106 per mouse). Splenic cells from the reconstituted (MIEV or PKC{alpha}-KR donor) mice or from an age-matched, unreconstituted RAG-1–/– (no donor) mouse were analyzed 3 weeks after injection. Flow cytometric analysis was carried out on GFP– and GFP+ populations, IgM-APC versus CD45R-PE, IgD-APC versus CD45-PE, and CD19-APC versus CD23-PE; percentages are indicated. Contour plots were live and size gated. Representative experiment of three separate adoptive transfers.

 
In addition to the generation of B-CLL-like populations within the spleen and lymph node of PKC{alpha}-KR-HPC-injected mice, we also noted a s.c. tumor cell mass at the site of injection. These tumors were only present in PKC{alpha}-KR-HPC-injected mice. Although tumors are not a characteristic of B-CLL, similar tumors develop when human B-CLL cells are injected s.c. into severe combined immunodeficient mice, suggesting that some PKC{alpha}-KR-expressing HPCs become trapped s.c. at the time of injection and develop into tumors (30). Flow cytometric analysis of the tumor mass revealed that the majority of the cells were GFP+ (89%; Fig. 6A) and that phenotypically the cells resembled the B-CLL population observed in the spleen and lymph node (IgMlo CD23+ CD5+ CD19hi IgDlo; Fig. 6A; data not shown). It has been shown previously that the level of gene expression in bicistronic retroviral vectors can be directly related to the intensity of GFP expression (Fig. 2A; refs. 21, 24); therefore, it is interesting to note that the expression levels of CD23, CD19, and CD5 were elevated with increasing GFP expression in the s.c. tumors. This was shown by comparing the MFI for specific cell surface markers on B lineage cells in PKC{alpha}-KR GFPlo compared with GFPhi populations: CD19 (98.0 versus 99.2), CD23 (90.1 versus 172), and CD5 (23.2 versus 66.7; Fig. 6A). Additionally and in agreement with our previous in vitro and in vivo data, IgM expression was down-regulated on the tumor cells with increasing GFP expression levels (16.2 versus 11.6; Fig. 6A). This finding suggests that the frequency of B-CLL cells increases with elevating levels of PKC{alpha}-KR expression in vivo (Fig. 6A).



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  		Figure 6. A tumor cell mass phenotypically resembling B-CLL forms s.c. on injection of RAG-1–/– mice with PKC{alpha}-KR-HPCs. HPC-enriched fetal liver cells were prepared from wild-type mice and retrovirally infected with PKC{alpha}-KR. A, retrovirally infected wild-type mouse-derived cells were injected i.p. into neonatal RAG-1–/– mice (1 x 106 per mouse). Analysis of the mice 3 weeks after injection revealed that GFP+ tumor cell masses developed s.c. at the point of injection. Flow cytometric analysis was carried out on GFPlow and GFPhi populations, IgM-APC versus CD45R-PE, IgD-APC versus CD45-PE, CD19-APC versus CD23-PE, and CD5-PE versus CD45R; percentages are indicated. All FACS plots were live and size gated. B, PI analysis was carried out on cell suspensions of either the freshly isolated tumor (day 0) or the tumor cells 5 days after they have been seeded onto OP9 cells in the presence of growth factors (day 5). Top, average ± SE percentage of cells at the indicated cell cycle stage; bottom, histogram of DNA content versus RCN. The cellular data were size gated.

 
This highly enriched population of B-CLL cells provided us with an ideal opportunity to analyze the cell cycle profile of in vivo–generated B-CLL cells. PI analysis revealed that tumor cells isolated fresh ex vivo were almost entirely arrested in the G0-G1 phase of the cell cycle (Fig. 6B), therefore bearing a striking similarity to the B-CLL cells isolated from humans (1, 28). Culture of these tumor-derived cells fresh ex vivo in the absence of both growth factors and stroma resulted in cell death, a finding that is common in B-CLL cells isolated from patients (data not shown). To determine whether the tumor cells could be induced to proliferate, therefore overcoming the cell cycle arrest, the tumor cells were seeded onto OP9 in the presence of growth factors and the cell cycle profile was determined after 5 days in culture. PI analysis revealed that the tumor cells reentered cell cycle (Fig. 6B), suggesting that although these cells were generated in vivo they maintain the ability to reenter the cell cycle when introduced into the proliferative conditions of the in vitro cocultures.

Collectively, our results establish that the disruption of PKC{alpha} signaling acts as an oncogenic trigger for developing B lymphocytes resulting in the generation of transformed cells that bear many of the key hallmarks of B-CLL both in vitro and in vivo.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article, we present novel data showing that attenuation of PKC{alpha} signaling in mouse HPCs results in the spontaneous generation of a population of cells that resembles human B-CLL at the level of (a) phenotype (CD19hi CD5+ CD23+ IgMlo), (b) cell cycle phase (halted in G0-G1 ex vivo), and (c) resistance to apoptosis (possibly due to an elevation in Bcl-2 expression). Moreover, we show that during the initiation of transformation PKC{alpha}-KR-expressing HPCs possess an enhanced proliferative capacity both in vitro and in vivo, potentially reflecting the dynamic cellular kinetics that exist during the progression of human B-CLL (31). Our data establish an important link between PKC{alpha} and B-CLL and provide an excellent model system to study the cellular and molecular mechanisms that drive the induction of B-CLL, therefore engendering the possibility of novel therapeutic avenues.

Whereas our results reveal that a B-CLL-like population of cells develops on stable expression of dominant-negative PKC{alpha}, studies using the PKC{alpha}–/– mouse model show that a lack of PKC{alpha} activity does not result in the development of spontaneous cancers, suggesting that PKC{alpha} does not normally behave as a tumor suppressor (32). This is in contrast to recent studies establishing that the Ras-GTPase-activating protein–associated docking proteins Dok-1 and Dok-2 act as tumor suppressors of chronic myelogenous leukemia (CML) as shown by the development of CML in Dok-1–/–Dok-2–/– mice (33, 34). In addition, reports establish that the signaling molecules SLP-65 (also known as BLNK/BASH) and Btk act as tumor suppressors in B lymphocytes as evidenced by the observation that SLP-65–/– and SLP-65–/–Btk–/– mouse models display a high incidence of pre-B-cell lymphomas (35–37). However, SLP-65–/– mouse-derived tumors require expression of the pre-BCR, whereas it seems that the induction of B-CLL by subversion of PKC{alpha} signaling can occur independently of pre-BCR expression. Indeed, a B-CLL-like population of cells can be generated on introduction of PKC{alpha}-KR into HPCs derived from RAG–/– mice, which lack the ability rearrange the heavy chain locus and are therefore blocked in development before the expression of a pre-BCR complex (20).4

Although knockout mouse models have highlighted the importance of PKCß and PKC{zeta} in controlling B-cell activation, survival, and proliferation (38–40) and PKC{delta} in mediating B-cell tolerance (41, 42), none of these models identify specific roles for individual PKC isoforms during early B-cell maturation, suggesting a functional redundancy exists between PKC family members (7). Interestingly, B lymphocytes from PKC{delta}–/– mice display splenomegaly and lymphadenopathy due to increases in the numbers of peripheral B cells (41, 42); therefore, it is perhaps surprising that HPCs expressing PKC{delta}-KR do not display a growth advantage in our B-cell generation systems. However, PKC{delta}–/– mouse-derived mature B cells proliferate because they are unable to undergo B-cell anergy in response to self-antigen (41, 42). Therefore, lack of proliferation observed in PKC{delta}-KR retrovirally infected cells may be due to an absence of antigen in our in vitro HPC-OP9 coculture system (43).

Our results indicate that retroviral infection of HPCs with a plasmid encoding dominant-negative PKC{alpha} induces the transformation of B lymphocytes in vivo and in vitro. The fact that our system revealed the transformation specifically of B lymphocytes is perhaps unsurprising, as our in vitro B-cell generation system uses the stromal cell line, OP9, derived from op/op mice, which are deficient in M-CSF (19). OP9 cells support hematopoiesis and lymphopoiesis from HSCs, but the lack of M-CSF expression contributes to the preferential development of B lymphocytes over macrophages (19). Our previous results suggest that expression of PKC{alpha}-KR does not trigger oncogenesis in developing T lymphocytes (21); however, it remains to be established whether subversion of PKC{alpha} signaling can generate an oncogenic signal in other hematopoietic cell lineages, such as natural killer lymphocytes, macrophages, or granulocytes, as has been established for several other cell types (14).

This is to our knowledge the first time that disruption of PKC{alpha} signaling has been specifically linked to the induction of B-CLL. Our studies show that the ability to induce the development of B-CLL in HPCs only occurs on PKC{alpha} but not PKC{delta} or PKC{zeta} subversion, suggesting that this may be a unique property of PKC{alpha}. PKC-mediated signaling pathways have been implicated previously in the maintenance of B-CLL cell survival as PKC inhibitors, and in some cases classic PKC inhibitors, are effective at inducing apoptosis of B-CLL cells, but the role of individual PKC isoforms has not been elucidated (3, 6, 43). At first sight, therefore, these studies seem to be at odds with our findings as we have clearly shown that the PKC{alpha}-KR-expressing B-CLL-like cells generated in our coculture system display a reduction in PKC activity and may indeed occur as a consequence of specifically targeting PKC{alpha} activity. However, this apparent discrepancy may simply reflect that we have used an established dominant-negative PKC{alpha} construct as opposed to using inhibitors that, at best, selectively inhibit classic PKC isoforms. Thus, these inhibitors might perhaps be targeting other PKC isoforms; alternatively, PKCß and/or PKC{gamma} could play a role dominant to that of PKC{alpha} in promoting B-CLL survival. Of note, PKC{alpha} and, more recently, Btk have been shown to function as adapter proteins in a manner that is independent of their kinase activity to transduce intracellular signals (44–47), suggesting that blocking the oncogenic property of PKC{alpha} is more complex than simply inhibiting its kinase activity. In conclusion, our studies reveal an important link between the subversion of PKC{alpha} signaling and the induction of B-CLL and have uncovered a potentially important avenue for developing drug therapies to treat of B-CLL.


   Acknowledgments
 
Grant support: Tenovus-Scotland and Medical Research Council, Medical Research Council career development award (A.M. Michie), and University of Glasgow medical faculty scholarship and Overseas Research Students award (R. Nakagawa).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank M.M. Harnett and S.M. Mason for their invaluable discussions and critical review of the article.


   Footnotes
 
4 Unpublished results. Back

Received 3/11/05. Revised 9/29/05. Accepted 10/18/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bannerji R, Byrd JC. Update on the biology of chronic lymphocytic leukemia. Curr Opin Oncol 2000;12:22–9.[CrossRef][Medline]
  2. Kay NE, Hamblin TJ, Jelinek DF, et al. Chronic lymphocytic leukemia. Hematology (Am Soc Hematol Educ Program) 2002;193–213.
  3. Barragán M, Bellosillo B, Campàs C, et al. Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells. Blood 2002;99:2969–76.[Abstract/Free Full Text]
  4. Cuni S, Perez-Aciego P, Perez-Chacon G, et al. A sustained activation of PI3K/NF-{kappa}B pathway is critical for the survival of chronic lymphocytic leukemia B cells. Leukemia 2004;18:1391–400.[CrossRef][Medline]
  5. Ringshausen I, Schneller F, Bogner C, et al. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase C{delta}. Blood 2002;100:3741–8.[Abstract/Free Full Text]
  6. Kitada S, Zapata JM, Andreeff M, Reed JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000;96:393–7.
  7. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 1995;9:484–96.[Abstract]
  8. Tan SL, Parker PJ. Emerging and diverse roles of protein kinase C in immune cell signalling. Biochem J 2003;376:545–52.[CrossRef][Medline]
  9. Izquierdo M, Downward J, Graves JD, Cantrell DA. Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells. Mol Cell Biol 1992;12:3305–12.[Abstract/Free Full Text]
  10. Downward J, Graves JD, Warne PH, et al. Stimulation of p21ras upon T-cell activation. Nature 1990;346:719–23.[CrossRef][Medline]
  11. Ruland J, Duncan GS, Elia A, et al. Bcl10 is a positive regulator of antigen receptor-induced activation of NF-{kappa}B and neural tube closure. Cell 2001;104:33–42.[CrossRef][Medline]
  12. Kolch W, Heidecker G, Kochs G, et al. Protein kinase Ca activates RAF-1 by direct phosphorylation. Nature 1993;364:249–52.[CrossRef][Medline]
  13. Dutil EM, Toker A, Newton AC. Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1). Curr Biol 1998;8:1366–75.[CrossRef][Medline]
  14. Michie AM, Nakagawa R. The link between PKCa regulation and cellular transformation. Immunol Lett 2005;96:155–62.[CrossRef][Medline]
  15. Yin JQ, Gao J, Shao R, et al. siRNA agents inhibit oncogene expression and attenuate human tumor cell growth. J Exp Ther Oncol 2003;3:194–204.[CrossRef][Medline]
  16. Tortora G, Ciardiello F. Antisense strategies targeting protein kinase C: preclinical and clinical development. Semin Oncol 2003;30:26–31.[Medline]
  17. Lahn M, Sundell K, Moore S. Targeting protein kinase C-a (PKC-a) in cancer with the phosphorothioate antisense oligonucleotide aprinocarsen. Ann N Y Acad Sci 2003;1002:263–70.[CrossRef][Medline]
  18. Soh JW, Lee EH, Prywes R, Weinstein IB. Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol Cell Biol 1999;19:1313–24.[Abstract/Free Full Text]
  19. Nakano T, Kodam H, Honjo T. Generation of lympho-hematopoietic cells from embryonic stem cells in culture. Science 1994;265:1098.[Abstract/Free Full Text]
  20. Mombaerts P, Iacomini J, Johnson RS, et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 1992;68:869–77.[CrossRef][Medline]
  21. Michie AM, Soh J-W, Hawley RG, et al. Allelic exclusion and differentiation by protein kinase C-mediated signals in immature thymocytes. Proc Natl Acad Sci U S A 2001;98:609.[Abstract/Free Full Text]
  22. Baier G. The PKC gene module: molecular biosystematics to resolve its T cell functions. Immunol Rev 2003;192:64–79.[CrossRef][Medline]
  23. Markowitz D, Goff S, Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 1988;62:1120–4.[Abstract/Free Full Text]
  24. Grady GC, Mason SM, Stephen J, et al. Cyclic adenosine 5'-monophosphate response element binding protein plays a central role in mediating proliferation and differentiation downstream of the pre-TCR complex in developing thymocytes. J Immunol 2004;173:1802–10.[Abstract/Free Full Text]
  25. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.[CrossRef][Medline]
  26. Noguchi P. Use of flow cytometry for DNA analysis. In: Coligan J, Kruisbeek A, Margulies D, Shevach E, Strober D, editors. Current protocols in immunology. New York: Green Publishing Associates; 1991. p. 5.7.1–5.7.4.
  27. Pennycook JL, Chang Y, Celler J, et al. High frequency of normal DJH joints in B cell progenitors in severe combined immunodeficiency mice. J Exp Med 1993;178:1007–16.[Abstract/Free Full Text]
  28. Reed JC. Molecular biology of chronic lymphocytic leukemia. Semin Oncol 1998;25:11–8.[Medline]
  29. Schimmer AD, Munk-Pedersen I, Minden MD, Reed JC. Bcl-2 and apoptosis in chronic lymphocytic leukemia. Curr Treat Options Oncol 2003;4:211–8.[Medline]
  30. Mohammad RM, Mohamed AN, Hamdan MY, et al. Establishment of a human B-CLL xenograft model: utility as a preclinical therapeutic model. Leukemia 1996;10:130–7.[Medline]
  31. Messmer BT, Messmer D, Allen SL, et al. In vivo measurements document the dynamic cellular kinetics of chronic lymphocytic leukemia B cells. J Clin Invest 2005;115:755–64.[CrossRef][Medline]
  32. Leitges M, Plomann M, Standaert ML, et al. Knockout of PKC{alpha} enhances insulin signaling through PI3K. Mol Endocrinol 2002;16:847–58.[Abstract/Free Full Text]
  33. Yasuda T, Shirakata M, Iwama A, et al. Role of Dok-1 and Dok-2 in myeloid homeostasis and suppression of leukemia. J Exp Med 2004;200:1681–7.[Abstract/Free Full Text]
  34. Niki M, Di Cristofano A, Zhao M, et al. Role of Dok-1 and Dok-2 in leukemia suppression. J Exp Med 2004;200:1689–95.[Abstract/Free Full Text]
  35. Flemming A, Brummer T, Reth M, Jumaa H. The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion. Nat Immunol 2003;4:38–43.[CrossRef][Medline]
  36. Jumaa H, Bossaller L, Portugal K, et al. Deficiency of the adaptor SLP-65 in pre-B-cell acute lymphoblastic leukaemia. Nature 2003;423:452–6.[CrossRef][Medline]
  37. Kersseboom R, Middendorp S, Dingjan GM, et al. Bruton's tyrosine kinase cooperates with the B cell linker protein SLP-65 as a tumor suppressor in Pre-B cells. J Exp Med 2003;198:91–8.[Abstract/Free Full Text]
  38. Leitges M, Schmedt C, Guinamard R, et al. Immunodeficiency in protein kinase cß-deficient mice. Science 1996;273:788–91.[Abstract]
  39. Kang SW, Wahl MI, Chu J, et al. PKCß modulates antigen receptor signaling via regulation of Btk membrane localization. EMBO J 2001;20:5692–702.[CrossRef][Medline]
  40. Martin P, Duran A, Minguet S, et al. Role of {zeta}PKC in B-cell signaling and function. EMBO J 2002;21:4049–57.[CrossRef][Medline]
  41. Mecklenbrauker I, Saijo K, Zheng N, et al. Protein kinase C{delta} controls self-antigen-induced B-cell tolerance. Nature 2002;416:860–5.[CrossRef][Medline]
  42. Miyamoto A, Nakayama K, Imaki H, et al. Increased proliferation of B cells and auto-immunity in mice lacking protein kinase C{delta}. Nature 2002;416:865–9.[CrossRef][Medline]
  43. Thomas A, Pepper C, Hoy T, Bentley P. Bryostatin induces protein kinase C modulation, Mcl-1 up-regulation and phosphorylation of Bcl-2 resulting in cellular differentiation and resistance to drug-induced apoptosis in B-cell chronic lymphocytic leukemia cells. Leuk Lymphoma 2004;45:997–1008.[CrossRef][Medline]
  44. Melendez AJ, Harnett MM, Allen JM. Crosstalk between ARF6 and protein kinase Ca in Fc{gamma}RI-mediated activation of phospholipase D1. Curr Biol 2001;11:869–74.[CrossRef][Medline]
  45. Mandil R, Ashkenazi E, Blass M, et al. Protein kinase Ca and protein kinase C{delta} play opposite roles in the proliferation and apoptosis of glioma cells. Cancer Res 2001;61:614612–9.
  46. Singer WD, Brown HA, Jiang X, Sternweis PC. Regulation of phospholipase D by protein kinase C is synergistic with ADP-ribosylation factor and independent of protein kinase activity. J Biol Chem 1996;271:4504–10.[Abstract/Free Full Text]
  47. Middendorp S, Zijlstra AJ, Kersseboom R, et al. Tumor suppressor function of Bruton tyrosine kinase is independent of its catalytic activity. Blood 2005;105:259–65.[Abstract/Free Full Text]



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